EP3204772B2 - Procédés permettant d'identifier des modulateurs de récepteurs de détection du calcium - Google Patents

Procédés permettant d'identifier des modulateurs de récepteurs de détection du calcium Download PDF

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EP3204772B2
EP3204772B2 EP15784884.7A EP15784884A EP3204772B2 EP 3204772 B2 EP3204772 B2 EP 3204772B2 EP 15784884 A EP15784884 A EP 15784884A EP 3204772 B2 EP3204772 B2 EP 3204772B2
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calcium
casr
sensing receptor
amino acid
receptor
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EP3204772A1 (fr
EP3204772B1 (fr
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Scott Joseph MCGRANE
Andrew John Taylor
Richard Masten FINE
Boris Klebansky
Matthew Ronald GIBBS
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Mars Inc
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Mars Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the presently disclosed subject matter relates to methods for identifying compounds that modulate the activity and/or expression of a calcium-sensing receptor.
  • Taste profiles for edible compositions include basic tastes such as sweet, salt, bitter, sour, umami and kokumi. Taste profiles have also been described as including free fatty acid tastes. Chemical compounds that elicit these tastes are often referred to as tastants. Without being bound by theory, it is hypothesized that tastants are sensed by taste receptors in the mouth and throat which transmit signals to the brain where the tastants and resulting taste profiles are registered.
  • Taste receptors include the calcium-sensing receptor (CaSR), which is a G-protein coupled receptor (GPCR) that detects changes in extracellular calcium levels and a close relative to the T1R1, T1R2 and T1R3 receptors, i.e., the sweet and umami receptors.
  • CaSR calcium-sensing receptor
  • GPCR G-protein coupled receptor
  • the calcium-sensing receptor has been shown to enhance sweet, salty and umami tastes, and function as a receptor for kokumi taste.
  • Pet food manufacturers have a long-standing desire to provide pet food products that have high nutritional value.
  • pet food manufacturers desire a high degree of palatability so that pets can receive the full nutritional benefit from their food.
  • domestic animals, especially cats are notoriously fickle in their food preferences, and often refuse to eat a pet food product that it has accepted over time or refuse to eat any more than a minimal amount of a pet food product.
  • This phenomenon may be, in part, due to the subtle differences in the sensory profiles of the raw material, which can be perceived by the domestic animals because of their gustatory and olfactory systems.
  • pet owners frequently change types and brands of pet food in order to maintain their pets in a healthy and contented condition.
  • the enhancement or modification can be to increase the intensity of a desirable attribute, to replace a desirable attribute not present or somehow lost in the pet food product, or to decrease the intensity of an undesirable attribute.
  • US 2012/0115176 describes a method for selecting compounds having a modulating effect on the activation state of a dimer of VFT-domain proteins.
  • the invention provides a method for identifying a composition for increasing palatability of a pet food product, comprising
  • a method for identifying compounds that enhance, increase and/or modulate the activity and/or expression of a calcium-sensing receptor comprises expressing a calcium-sensing receptor having a nucleotide sequence set forth in SEQ ID NO: 1, 2, or 7, or a fragment or variant thereof, in a cell.
  • the method can further comprise contacting the cell expressing the calcium-sensing receptor with a test compound and determining the activity and/or expression of the calcium-sensing receptor in the presence of the compound as compared to the activity and/or expression of the receptor in the absence of the compound.
  • a method for identifying compounds that enhance, increase and/or modulate the activity of a calcium-sensing receptor comprises expressing a calcium-sensing receptor having an amino acid sequence set forth in SEQ ID NO: 4, or 5, or a fragment or variant thereof, in a cell.
  • the method can further comprise contacting the cell expressing the calcium-sensing receptor with a test compound and determining the activity and/or expression of the calcium-sensing receptor in the presence of the compound as compared to the activity and/or expression of the receptor in the absence of the compound, as defined in the claims.
  • the present disclosure provides a method for identifying a composition that modulates the activity of a calcium-sensing receptor (CaSR) comprising (a) contacting a test agent with a CaSR, (b) detecting an interaction between the test agent and one or more amino acids in a Venus Flytrap domain (VFT) or 7 transmembrane domain (7TM) of the CaSR, and (c) selecting as the composition, a test agent that interacts with one or more of the amino acids, as further defined in the claims.
  • CaSR calcium-sensing receptor
  • the present disclosure provides a method for identifying a composition that modulates the activity of a calcium-sensing receptor (CaSR) comprising (a) contacting a CaSR agonist with a CaSR, (b) determining the activity of the CaSR, (c) contacting a test agent with the CaSR, (d) determining the activity of the CaSR, and (e) selecting the test agent as the composition when the activity of (d) is greater than the activity of (b), as further defined in the claims.
  • CaSR calcium-sensing receptor
  • aroma and “smell” refer to an olfactory response to a stimulus.
  • an aroma can be produced by aromatic substances that are perceived by the odor receptors of the olfactory system.
  • the terms “modulates” or “modifies” refers an increase or decrease in the amount, quality or effect of a particular activity of a receptor and/or an increase or decrease in the expression, activity or function of a receptor.
  • Modules refer to any inhibitory or activating compounds identified using in silico, in vitro and/or in vivo assays for, e.g., agonists, antagonists and their homologs, including fragments, variants and mimetics.
  • Inducers refer to modulating compounds that increase, induce, stimulate, open, activate, facilitate, enhance activation, sensitize or upregulate a receptor or pathway of interest.
  • operably linked when applied to DNA sequences, e.g., in an expression vector, indicates that the sequences are arranged so that they function cooperatively in order to achieve their intended purposes, i.e., a promoter sequence allows for initiation of transcription that proceeds through a linked coding sequence as far as the termination signal.
  • Amino acid analogs and derivatives can refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group and an R group, e.g., homoserine, norleucine, methionine sulfoxide and methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics means chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • isolated refers to a nucleic acid, a polypeptide, or other biological moiety that is removed from components with which it is naturally associated.
  • isolated can refer to a polypeptide that is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macromolecules of the same type.
  • isolated with respect to a polynucleotide can refer to a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
  • the term "recombinant" can be used to describe a nucleic acid molecule and refers to a polynucleotide of genomic, RNA, DNA, cDNA, viral, semisynthetic or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of polynucleotide with which it is associated in nature.
  • fusion refers to joining of different peptide or protein segments by genetic or chemical methods wherein the joined ends of peptide or protein segments may be directly adjacent to each other or may be separated by linker or spacer moieties such as amino acid residues or other linking groups.
  • the presently disclosed subject matter provides calcium-sensing receptors (CaSRs) for use in the disclosed methods.
  • the calcium-sensing receptors of the present disclosure can include mammalian calcium-sensing receptors such as, but not limited to, felines, canines and humans.
  • a calcium-sensing receptor for use in the presently disclosed subject matter encompasses a feline calcium-sensing receptor having the nucleotide sequence set forth in SEQ ID NO: 1 or 7 and/or the amino acid sequence set forth in SEQ ID NO:4, including fragments thereof (e.g., functional fragments thereof) and variants thereof.
  • a calcium-sensing receptor for use in the presently disclosed subject matter encompasses a human calcium-sensing receptor having the nucleotide sequence set forth in SEQ ID NO: 3 and/or the amino acid sequence set forth in SEQ ID NO:6, including fragments thereof ( e.g., functional fragments thereof) and variants thereof.
  • the percent identity of two amino acid sequences or of two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions.
  • the determination of percent identity between two sequences can be determined using a mathematical algorithm known to those of skill in the art.
  • a non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268 , modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877 .
  • the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402 .
  • PSI-Blast can be used to perform an iterated search, which detects distant relationships between molecules.
  • the disclosed subject matter provides for the use of an isolated or purified calcium-sensing receptor and/or variants and fragments thereof.
  • the disclosed subject matter also encompasses the use of sequence variants.
  • variation can occur in either or both the coding and non-coding regions of a nucleotide sequence of a calcium-sensing receptor.
  • Variants can include a substantially homologous protein encoded by the same genetic locus in an organism, i.e., an allelic variant.
  • Variants also encompass proteins derived from other genetic loci in an organism, e.g., feline, but having substantial homology to the calcium-sensing receptor, i.e., a homolog.
  • Variants can also include proteins substantially homologous to the calcium-sensing receptor but derived from another organism, i.e., an ortholog. Variants also include proteins that are substantially homologous to the calcium-sensing receptor that are produced by chemical synthesis. Variants also include proteins that are substantially homologous to the calcium-sensing receptor that are produced by recombinant methods.
  • Orthologs, homologs and allelic variants can be identified using methods well known in the art. These variants can include a nucleotide sequence encoding a receptor that is at least about 60-65%, about 65-70%, about 70-75, about 80-85%, about 90-95%, about 95-99% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1, 2, 3 or 7, or fragments thereof. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ ID NO: 1, 2, 3 or 7, or a fragment thereof.
  • two polypeptides are substantially homologous when the amino acid sequences are at least about 60-65%, about 65-70%, about 70-75, about 80-85%, about 90-95%, about 95-99% or more homologous to the amino acid sequences shown in SEQ ID NO: 4, or 5, or a fragment thereof, as defined in the claims.
  • a substantially homologous amino acid sequence, according to the disclosed subject matter will be encoded by a nucleic acid sequence hybridizing to the nucleic acid sequence, or portion thereof, of the nucleotide sequence shown in SEQ ID NOs: 1, 2, 3 or 7 under stringent conditions.
  • the calcium-sensing receptors for use in the methods of the disclosed subject matter include calcium-sensing receptors having additions, deletions or substitutions of amino acid residues (variants) which do not substantially alter the biological activity of the receptor.
  • Those individual sites or regions of the calcium-sensing receptors which may be altered without affecting biological activity can be determined by examination of the structure of the calcium-sensing receptor extracellular domain, for example.
  • alanine scanning mutagenesis selected amino acid residues are individually substituted with a neutral amino acid (e.g., alanine) in order to determine the effects on biological activity.
  • one or more amino acid residues within a calcium-sensing receptor can be replaced with other amino acid residues from the same side chain family and the altered protein can be tested for retained function using the functional assays described herein.
  • Modifications can be introduced into a calcium-sensing receptor of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. If such substitutions result in a retention in biological activity, then more substantial changes can be introduced and/or other additions/deletions may be made and the resulting products screened.
  • deletions or additions can be from 5-10 residues, alternatively from 2-5 amino acid residues or from 1-2 residues.
  • fusion proteins that comprise a calcium-sensing receptor, or fragment thereof.
  • the disclosed subject matter provides for fusion proteins of a calcium-sensing receptor, or functional fragments thereof, and an immunoglobulin heavy chain constant region.
  • a fusion protein of the present disclosure can include a detectable marker, a functional group such as a carrier, a label, a stabilizing sequence or a mechanism by which calcium-sensing receptor agonist binding can be detected non-limiting examples of a label include a FLAG tag, a His tag, a MYC tag, a maltose binding protein and others known in the art, as defined in the claims.
  • the presently disclosed subject matter also provides nucleic acids encoding such fusion proteins, vectors containing fusion protein-encoding nucleic acids and host cells comprising such nucleic acids or vectors.
  • fusions can be made at the amino terminus (N-terminus) of a calcium-sensing receptor or at the carboxy terminus (C-terminus) of a calcium-sensing receptor.
  • the calcium-sensing receptors disclosed herein can contain additional amino acids at the N-terminus and/or at the C-terminus end of the sequences, e.g ., when used in the methods of the disclosed subject matter.
  • the additional amino acids can assist with immobilizing the polypeptide for screening purposes, or allow the polypeptide to be part of a fusion protein, as disclosed above, for ease of detection of biological activity.
  • the present disclosure further provides methods for identifying compounds that modulate the activity and/or expression of a calcium-sensing receptor, as defined in the claims.
  • the modulator can be an agonist or an antagonist.
  • the presently disclosed subject matter provides in silico and in vitro methods for identifying compounds that modulate the activity and/or expression of a calcium-sensing receptor, disclosed above.
  • the presently disclosed subject matter further provides in silico methods for identifying compounds that can potentially interact with a calcium-sensing receptor and/or modulate the activity and/or expression of a calcium-sensing receptor, as defined in the claims.
  • the method can include predicting the three-dimensional structure (3D) of a calcium-sensing receptor and screening the predicted 3D structure with putative calcium-sensing receptor modulating compounds (i.e., test compounds).
  • the method can further include predicting whether the putative compound would interact with the binding site of the receptor by analyzing the potential interactions with the putative compound and the amino acids of the receptor.
  • the method can further include identifying a test compound that can bind to and/or modulate the biological activity of the calcium-sensing receptor by determining whether the 3D structure of the compound fits within the binding site of the 3D structure of the receptor.
  • the calcium-sensing receptor for use in the disclosed method can have the amino acid sequence of SEQ ID NO: 4, or 5.
  • the calcium-sensing receptor for use in the presently disclosed subject matter can include a receptor comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 4, or 5.
  • the calcium-sensing receptor for use in the disclosed method can have the nucleotide sequence of SEQ ID NO: 1, 2, or 7, or a fragment or variant thereof.
  • the calcium-sensing receptor for use in the presently disclosed subject matter can include a receptor comprising a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, 2, or 7, or a fragment or variant thereof, as defined in the claims.
  • Non-limiting examples of compounds e.g., potential calcium-sensing receptor modulators
  • the test compound can be a small chemical molecule.
  • structural models of a calcium-sensing receptor can be built using crystal structures of other GPCRs as templates for homology modelling. For example, and not by way of limitation, structural models can be generated using the crystal structures of Group C GPCRs.
  • a structural model of a calcium-sensing receptor can be based on a known or a combination of known crystal structures of GPCRs. ( See, e.g., Lee et al., Eur J Pharmacol. 2015 May 14. pii: S0014-2999(15)30012-1 ).
  • a structural model of a calcium-sensing receptor can be generated based on the crystal structure of an mGluR protein.
  • a structural model of the flytrap domain (VFT) of a calcium-sensing receptor can be generated based on the crystal structure having the protein data base (PDB) ID No. 1EWK.
  • a structural model of the 7 transmembrane domain (7TM) of a calcium-sensing receptor can be generated based on the crystal structures of mGluR proteins having PDB ID Nos. 4OR2 and 4OO9.
  • Figure 13 depict structural models of calcium-sensing receptors that can be used in the disclosed in silico methods. Any suitable modeling software known in the art can be used.
  • the Modeller software package can be used to generate the three-dimensional protein structure.
  • the in silico methods of identifying a compound that binds to a calcium-sensing receptor comprises determining whether a test compound interacts with one or more amino acids of a calcium-sensing receptor interacting domain, as described herein.
  • the compounds identified according to the methods described herein that modulate the activity of a calcium-sensing receptor interact with one or more amino acids in a transmembrane domain of the calcium-sensing receptor, for example, a seven transmembrane domain (7TM).
  • the amino acids that the compounds interact with comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of ARG680; PHE684; GLY685; PHE688; VAL689 on Helix 3; GLN735 on Helix 4; ALA772; PHE775; LEU776; THR780; CYS781 on Helix 5; PHE814; VAL817; TRP818; PHE821 on Helix 6; GLU837; ALA840; ILE841; ALA844 on Helix 7; and MET771 and GLU767 on the EC2 loop of a calcium-sensing receptor, for example, a calcium-sensing receptor comprising a feline calcium-sensing receptor described by SEQ ID NO:4, or a canine calcium-sensing receptor described by SEQ ID NO:5.
  • a calcium-sensing receptor comprising a feline calcium-sensing receptor described by
  • the method for identifying compounds that modulate the activity and/or expression of a calcium-sensing receptor comprises measuring the biological activity of a calcium-sensing receptor in the absence and/or presence of a test compound, as defined in the claims.
  • the method can include measuring the biological activity of a calcium-sensing receptor in the presence of varying concentrations of the test compound.
  • the method can further include identifying the test compounds that result in a modulation of the activity and/or expression of the calcium-sensing receptor compared to the activity and/or expression of the calcium-sensing receptor in the absence of the test compound.
  • the compounds identified according to the methods described herein increase the biological activity of a calcium-sensing receptor by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, compared to the biological activity of the calcium-sensing receptor when the compound is not present.
  • the method can further include analyzing two or more, three or more or four or more test compounds in combination.
  • the two or more, three or more or four or more test compounds can be from different classes of compounds, e.g ., amino acids and small chemical compounds.
  • the method can include analyzing the effect of one or more small chemical test compounds on the biological activity and/or expression of a calcium-sensing receptor in the presence of one or more amino acid test compounds.
  • the method for identifying compounds that modulate the activity and/or expression of a calcium-sensing receptor comprises expressing a calcium-sensing receptor in a cell line and measuring the biological activity of the receptor in the presence and/or absence of a test compound.
  • the method can further comprise identifying test compounds that modulate the activity of the receptor by determining if there is a difference in receptor activation in the presence of a test compound compared to the activity of the receptor in the absence of the test compound.
  • the selectivity of the putative calcium-sensing receptor modulator can be evaluated by comparing its effects on other GPCRs or taste receptors, e.g., umami, fatty acid, T1R, etc. receptors.
  • the activity of the calcium-sensing receptor can be determined by the detection of secondary messengers such as, but not limited to, cAMP, cGMP, IP3, DAG or calcium.
  • the activity of the calcium-sensing receptor can be determined by the detection of the intracellular calcium levels. Monitoring can be by way of luminescence or fluorescence detection, such as by a calcium sensitive fluorescent dye.
  • the intracellular calcium levels can be determined using a cellular dye, e.g., a fluorescent calcium indicator such as Calcium 4.
  • the intracellular calcium levels can be determined by measuring the level of calcium binding to a calcium-binding protein, for example, calmodulin.
  • activity of the calcium-sensing receptor can be determined by detection of the phosphorylation, transcript levels and/or protein levels of one or more downstream protein targets of the calcium-sensing receptor.
  • the cell line used in the disclosed methods can include any cell type that is capable of expressing a calcium-sensing receptor.
  • Non-limiting examples of cells that can be used in the disclosed methods include HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), Xenopus oocytes, HEK-293 cells and murine 3T3 fibroblasts.
  • the method can include expressing a calcium-sensing receptor in HEK-293 cells.
  • the method can include expressing a calcium-sensing receptor in COS cells.
  • the cells constitutively express the calcium-sensing receptor.
  • expression of the CaSR by the cells is inducible.
  • the introduction of a nucleic acid into a cell can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993 ); Cohen et al., Meth. Enzymol. 217:618-644 (1993 ); Cline, Pharmac. Ther.
  • the nucleic acid encoding a calcium-sensing receptor is comprised in a cloning vector, for example, a pcDNA3.1 vector or a pcDNA5 TO vector, that is introduced into the cell.
  • the in vitro assay comprises cells expressing a calcium-sensing receptor that is native to the cells.
  • examples of such cells expressing a native calcium-sensing receptor include, for example but not limited to, dog (canine) and/or cat (feline) taste cells (e.g., primary taste receptor cells).
  • the dog and/or cat taste cells expressing a calcium-sensing receptor are isolated from a dog and/or cat and cultured in vitro.
  • the taste receptor cells can be immortalized, for example, such that the cells isolated from a dog and/or cat can be propagated in culture.
  • expression of a calcium-sensing receptor in a cell can be induced through gene editing, for example, through use of the CRISPR gene editing system to incorporate a calcium-sensing receptor gene into the genome of a cell, or to edit or modify a calcium-sensing receptor gene native to the cell.
  • compounds identified as modulators of a calcium-sensing receptor can be further tested in other analytical methods including, but not limited to, in vivo assays, to confirm or quantitate their modulating activity.
  • methods described herein can comprise determining whether the calcium-sensing receptor modulator is a kokumi taste enhancing compound, e.g., a calcium-sensing receptor agonist.
  • the methods of identifying a calcium-sensing receptor modulator can comprise determining whether a test compound modulates the activity of the receptor when the receptor is contacted with an agonist, or whether the test compound can modulate the activity of a positive allosteric modulator (PAM).
  • Test compounds that increase or decrease the effect of said agonist or PAM on the receptor can be selected as a calcium-sensing receptor modulating compound (e.g., as an allosteric modulator).
  • the calcium-sensing receptor modulators of the present application are identified through in silico modeling of a calcium-sensing receptor ("Kokumi receptor"), a feline or a canine calcium-sensing receptor as defined in the claims, wherein the calcium-sensing receptor agonists of the present application comprise a structure that fits within a binding site of the calcium-sensing receptor.
  • the in silico method comprises the in silico methods described above and in the Examples section of the present application.
  • the calcium-sensing receptor modulators of the present application are identified through an in vitro method, wherein the calcium-sensing receptor agonist compounds activate and/or modulate a calcium-sensing receptor, disclosed herein, expressed by cells in vitro.
  • the in vitro method comprises the in vitro methods described above and in the Examples section of the present application.
  • a homology model of the feline and canine CaSR 7M domain was generated based on the crystal structures of 4OR2 and 4OO9 from PDB ( see helix plot of Figure 14 ; see also, Figures 14 , 15 and 16 for helix plots of canine, feline and human CaSRs, respectively).
  • 4OR2 is the crystal structure of the transmembrane domain of mGluR1 from Group C GPCR bound to a negative allosteric modulator (NAM) ( see Wu et. al., Science, 344(6179):58-64 (2014 )).
  • Calindol was observed to have the following potential interactions with the amino acids of allosteric site of human, canine and feline CaSR: ARG680; PHE684; GLY685; PHE688; VAL689 on Helix 3; GLN735 on Helix 4; ALA772; PHE775; LEU776; THR780; CYS781 on Helix 5; PHE814; VAL817; TRP818; PHE821 on Helix 6; GLU837; ALA840; ILE841; ALA844 on Helix 7; and MET771 and GLU767 on the EC2 loop of the CaSR.
  • HEK293 cells that stably express a calcium-sensing receptor are exposed to putative compounds to modulate the activity and/or expression of the calcium-sensing receptor.
  • Activation of the calcium-sensing receptor is detected by a change in intracellular calcium levels using a calcium sensitive fluorescent dye.
  • Cells that do not express the calcium-sensing receptor or that are not contacted with the compound are used as a control.
  • a FLIPR ® Tetra or a FlexStation ® 3 is used for data capture.
  • the present example describes an in vitro assay for identifying compounds that modulate the activation of the canine calcium-sensing receptor.
  • HEK293 cells that stably express the canine CaSR are exposed to putative compounds to modulate the activity and/or expression of the calcium-sensing receptor.
  • Activation of the calcium-sensing receptor is detected by a change in intracellular calcium levels using a calcium sensitive fluorescent dye.
  • Cells that do not express the calcium-sensing receptor or that are not contacted with the compound are used as a control.
  • a FLIPR ® Tetra or a FlexStation ® 3 is used for data capture.
  • EC 50 half maximal effective concentration
  • the present example describes an in vitro assay for identifying compounds that modulate the activation of the feline calcium-sensing receptor.
  • the full length coding sequence of the calcium-sensing receptor (CaSR, a GPCR (3, C or Glutamate family), naturally coupled to Gaq/11 and Gai (ligand-directed signaling)) from Felis catus (fCaSR) having the sequence described by SEQ ID NO: 7 was synthesized and sub-cloned into suitable expression vectors.
  • the cat CaSR expression constructs were stably transfected in two mammalian cell lines, the HEK-natClytin and the HEK T-Rex/natClytin, two cell lines that allow for detection of changes in intracellular calcium levels through luminescence.
  • a pure selected clone (K 5.1) was used to analyze 12 CaSR ligands (including calcium as a reference agonist control) in a high-throughput screening (HTS) multi-plate test for agonist and positive allosteric modulator (PAM) effects. 7 of the ligands tested (including calcium control) were identified as agonists, and 2 were identified as PAM of CaSR, demonstrating that the in vitro cellular assay described by the present study is a reliable, reproducible and robust functional cell based assay for the cat CaSR receptor suitable for HTS.
  • HTS high-throughput screening
  • PAM positive allosteric modulator
  • HEK-natClytin and HEK T-Rex/natClytin cells were transfected with vectors comprising fCaSR. Mock transfections were carried out in parallel as negative controls. After antibiotic selections, a 1° limiting dilution of the two transfected target and mock pools were performed and then analyzed with calcium as a reference agonist and luminescence photoprotein as read out of activated fCaSR using a FLIPR ® Tetra screening system. Based on clone pool analysis results, the HEK T-Rex/natClytin responding clones were selected for further evaluation. A 2° limiting dilution of selected clones was performed.
  • K 5.1 A final pure selected clone (K 5.1) was analyzed in a HTS multi-plate test for agonist or positive allosteric modulator (PAM) effects of 12 CaSR ligands (including calcium as a reference agonist control). 10.000 cw of K 5.1 were seeded on poly-D-lys coated 384 well MTPs in medium containing 1 ⁇ g/mL of Doxycycline. Screening with the 12 ligands (including calcium control) was conducted 24 hours later. Dose response curves for the activation of CaSR by the ligands in agonist mode testing are shown in Figure 18 .
  • Dose response curves for the activation of CaSR by the ligands cinacalcet and calindol in PAM mode testing are shown in Figure 19 .
  • Table 2 7 of the ligands tested (including calcium control) activated CaSR as agonists, and 2 (cinacalcet and calindol) activated CaSR as PAMs.
  • the ECso value of the ligand was determined.
  • the term half maximal effective concentration (ECso) refers to the concentration of a compound which induces a response halfway between the baseline and the maximum after a specified exposure time. Table 2.
  • clone K4 After restriction analysis with BamHI enzyme for the screening of positive constructs, clone K4, with correct DNA fragmentation (6217 + 2781 pb), was selected. The presence of the entire fCaSR coding region was confirmed by sequencing.
  • the pcDNA3.1_fCaSR vector clone comprising the fCaSR coding region is shown in Figure 20 .
  • Transfection of HEK-natClytin and HEK-293 T-REx cell lines was performed by electroporation. 2x10 6 cells detached from 60-70% confluent flasks were transfected with a total amount of 10 ⁇ g of DNA construct by using the Gene Pulser II electroporator (Biorad) (parameters: 300 Volts, 950 ⁇ F). Transfected cells were immediately diluted in wild-type complete medium and seeded in T75 flasks. After 48h, the proper antibiotic concentration was added to the medium and cells were cultured at 37°C 5% CO 2 for about 3 weeks to generate stable pools.
  • Biorad Gene Pulser II electroporator
  • Culture medium HEK-293 T-REx cell line.
  • DMEM High Glucose (Lonza BioWhittaker cat. BE12-604F/U1; 500 mL) supplemented with Fetal Bovine Serum TET-FREE (Euroclone cat. EC S0182L; 50 mL), Penicillin-Streptomycin (BioWhittaker, cat. DE17-602E; 5 mL of 100x Solution), Blasticidin 5 ⁇ g/ml (InvivoGen, cat. ant-bl-1).
  • HEK T-Rex/natClytin cell medium was supplemented with 1 ⁇ g/mL of Puromicin (InvivoGen cat. ant-pr-1).

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Claims (9)

  1. Méthode d'identification d'une composition destinée à augmenter la palatabilité d'un produit alimentaire pour animaux familiers, comprenant
    (a) la mise en contact d'un agent de test avec un récepteur-détecteur de calcium (CaSR) à l'aide d'une modélisation in silico,
    (b) la détection d'une interaction in silico entre l'agent de test et un ou plusieurs acides aminés dans un domaine de Dionée attrape-mouche (VFT) ou un domaine transmembranaire 7 (7TM) du CaSR, et
    (c) la sélection, en tant que la composition, d'un agent de test qui interagit avec un ou plusieurs des acides aminés,
    dans laquelle le CaSR est choisi dans le groupe constitué par un CaSR félin comprenant une séquence d'acides aminés au moins 85 % identique à la séquence d'acides aminés de SEQ ID n° 4 et un CaSR canin comprenant une séquence d'acides aminés au moins 85 % identique à la séquence d'acides aminés de SEQ ID n° 5,
    dans laquelle les un ou plusieurs acides aminés dans le VFT sont choisis dans le groupe constitué par ASN64, PHE65, ASN102, THR145, SER169, SER170, ASP190, GLN193, ASP216, TYR218, SER271, SER272, GLY273, GLU297, ALA298, TRP299, ALA300, SER301, SER302, LEU304, ALA321, TYR411, THR412 et HIS413 ;
    dans laquelle les un ou plusieurs acides aminés dans le 7TM sont choisis dans le groupe constitué par ARG680 ; PHE684 ; GLY685 ; PHE688 ; VAL689 sur l'Hélice 3 ; GLN735 sur l'Hélice 4 ; ALA772 ; PHE775 ; LEU776 ; THR780 ; CYS781 sur l'Hélice 5 ; PHE814 ; VAL817 ; TRP818 ; PHE821 sur l'Hélice 6 ; GLU837 ; ALA840 ; ILE841 ; ALA844 sur l'Hélice 7 ; MET771 et GLU767 sur la boucle EC2 ; et
    en outre dans laquelle un dosage in vitro est effectué afin de déterminer si l'agent de test qui interagit avec un ou plusieurs des acides aminés est un modulateur de CaSR.
  2. Méthode selon la revendication 1, la méthode comprenant la détection d'une interaction entre l'agent de test et un ou plusieurs acides aminés dans une région charnière ou une région de chaîne principale du VFT.
  3. Méthode selon la revendication 1, dans laquelle l'interaction a lieu entre l'agent de test et un ou plusieurs acides aminés dans le VFT.
  4. Méthode selon la revendication 1, dans laquelle l'interaction de méthode a lieu entre l'agent de test et un ou plusieurs acides aminés dans le 7TM.
  5. Méthode selon la revendication 1, dans laquelle l'étape (c) comprend en outre la sélection, en tant que la composition, d'un agent de test qui augmente l'activité du CaSR.
  6. Méthode selon l'une quelconque des revendications 1-5, dans laquelle le dosage est un dosage in vitro qui comprend la comparaison de l'effet de l'agent de test à un agoniste de CaSR.
  7. Méthode selon l'une quelconque des revendications 1-5, dans laquelle le dosage est un dosage in vitro comprenant :
    (a) la mise en contact d'un agoniste de CaSR avec un récepteur-détecteur de calcium (CaSR),
    (b) la détermination de l'activité du CaSR,
    (c) la mise en contact de l'agent de test avec le CaSR,
    (d) la détermination de l'activité du CaSR, et
    (e) la sélection de l'agent de test en tant que la composition lorsque l'activité de (d) est supérieure à l'activité de (b),
    dans laquelle le CaSR est choisi dans le groupe constitué par un CaSR félin comprenant une séquence d'acides aminés au moins 85 % identique à la séquence d'acides aminés de SEQ ID n° 4 et un CaSR canin comprenant une séquence d'acides aminés au moins 85 % identique à la séquence d'acides aminés de SEQ ID n° 5.
  8. Méthode selon une quelconque revendication précédente, dans laquelle le CaSR est exprimé par une cellule, et dans laquelle l'agent de test est mis en contact avec la cellule.
  9. Méthode selon la revendication 8, dans laquelle la cellule exprime une photoprotéine fixant le calcium.
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CN109996453A (zh) * 2016-04-14 2019-07-09 马斯公司 用于调节厚味味道的调节钙敏感受体活性的化合物和含有该化合物的宠物食品
CN110656088A (zh) * 2018-06-29 2020-01-07 华中科技大学 一种稳定表达人源CaSR基因的细胞模型及其构建方法
EP3934446A4 (fr) * 2019-03-05 2023-05-03 Mars Incorporated Peptides modulant l'activité du récepteur de détection du calcium pour moduler le goût kokumi et produits alimentaires pour animaux de compagnie les contenant

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Summary document showing publicly available crystal structures of class A G protein-coupled receptors and class C G protein-coupledreceptors at priority date of D5 and the patent respectively †
Summary of amendments made to claim 1 of each Auxiliary Request †
Updated version of D14 †

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